The average lifespan of human being approaches 100 by virtue of improvement of the 21st century medical development and the quality of life. As the lifespan of human being prolongs, people are more interested in preventing diseases and anti-aging. Wrinkles and freckles in the skin increase and skin-regeneration decrease due to external stimuli and body skin aging, as times go by. Through those signs, the body aging can be confirmed with eyes, and people are continuously and gradually interested in preventing the skin aging and use therapeutic agents and cosmetics for preventing a variety of skin aging. To meet the interests, cosmetic companies continuously search for novel substances.
For existing whitening and anti-aging effective substances, arbutin, ascorbic acid derivatives selina, etc., are used.
Skin pigmentation occurs in association with the production of melanin in melanocytes. The production of melanin increases due to external stimuli (UV, inflammation) and active oxygen in the body. The melanin production pathway is as follows: tyrosine is oxidized to dopa, then to dopaquinone, and becomes 5,6-dihydroxyindole-2-carboxylic acid by dopachrome, and then a final eumelanin is produced. Tyrosinase, which is involved in the pathway of the oxidation of tyrosine to dopaquinone and the formation of 5,6-dihydroxyindole-2-carboxylic acid, is the most important enzyme for the production of melanin. Thus, the inhibition of tyrosinase is a main target in searching for substances with whitening effects.
Anti-aging associated substances include retinoid, silicic acid, mevalonolactone (mevalonic acid, MA), adenosine, retinyl palmitate, etc. They have effects of regulating skin cell regeneration, collagen production regulation, wrinkle improvement, etc. In order to prevent skin aging, the activation of skin cell regeneration, promotion of collagen formation, antioxidant activity, etc., are necessary. The skin is composed of the epidermis, dermis, and subcutaneous tissue, and fibroblast in the dermis significantly affects skin aging. Cellular aging resulting from the reduction of the number of fibroblasts causes the damage on skin tissues.
Also, in skin wound repair processes, the migration and proliferation of fibroblasts and wound contraction are important. Fibroblasts are involved in collagen production, and 90% of the dermis is composed of collagen. Collagen affects skin moisturizing and elasticity.
The regulation of inflammatory response is closely related to causes of inflammation-associated diseases, and the inflammatory response is associated with various signal transduction, for example, sequential activation of cyclooxygenase, NO synthetase, cytokine, etc. The excessive production of inflammatory mediators including NO, interleukin such as IL-6 and IL-1β, and TNF-α mediates inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel diseases, osteoporosis, psoriasis, endotoxemia, toxic shock syndrome, etc. Inflammatory response occurring in human bodies can be broadly classified as acute inflammatory response and chronic inflammatory response. Acute inflammatory response is the response quickly occurring in order to protect the body from antigen introduced from the outside. The initialization of acute inflammation accompanies the secretion of inflammatory cytokines, such as interleukin-1 beta, interleukin-6, tumor necrosis factor-alpha (TNF-α), etc., and the increase of synthesis genes of inflammatory mediators, such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), etc. Macrophages activated by the secreted inflammation regulatory substances effectively remove introduced antigen, and once the antigen is completely removed, no further progress occurs. Thus, it is rare that acute inflammatory response leads to human diseases. However, in the case the acute inflammatory response develops into certain chronic inflammatory diseases, such as pertussis, the expression of IL-23 increases, and it develops into chronic inflammation (Infect. Immun., 73: 1590-1597, 2005). Unlike acute inflammatory response, chronic inflammatory response is the prolonged inflammatory response caused by internal causes in the body, rather than by external antigen, and results from in the increase of the expression of IL-17 and IL-23 due to inflammatory response including macrophages, neutrophils, and T lymphocytes, not simple inflammatory response by macrophages, and thereby the continuous expression of inflammatory cytokines such as TNF-α (Trends in Immunol, 27: 17-23, 2006) occurs. For the treatment of chronic inflammatory diseases, conventionally, the following drugs are most commonly used: anti-inflammatory drugs by steroids such as prednisolone, nonsteroidal anti-inflammatory drugs (NSAID) such as naproxen, and immunosuppressant drugs which combine with calcineurin such as cyclosporine or FK506, which is a calcium and calmodulin dependent protein phosphatase, to suppress the activity. However, these immunosuppressant drugs including steroids entail side effects, such as nephrotoxicity, inflammation, lymphoma, diabetes, tremor, headache, diarrhea, hypertension, nausea, renal dysfunction, etc. Thus, it is urgent to develop effective therapeutic agents for inflammatory diseases suppressing excessive expression of inflammatory mediators, while reducing side effects of the currently used therapeutic agents for inflammatory disease.
Further, the inflammatory response refers to a series of complex biological response such as secretion of inflammatory mediators activating enzymes, body fluid infiltration, cell migration, tissue damage, etc., which are associated with various inflammatory mediators and immune cells in topical blood vessels and body fluids when tissues (cells) are damaged or infected by external sources of inflammation (bacteria, viruses, fungi, various types of substances causing allergies), and external signs such as redness, swelling, heat, pain, etc. In normal cases, inflammatory response removes the external source of inflammation and regenerates damaged tissues, so as to repair the functions of living things. However, if antigen is not removed or inflammatory response excessively or continuously occurs because of internal substances, damage on mucous membranes is promoted, which leads to some diseases, such as cancer. As causes of inflammation production in the body, various biochemical phenomena are involved in inflammation, and particularly, it is known that nitric oxide synthase (NOS) generating nitric oxide (NO) and enzymes associated with biosynthesis of prostaglandin play an important role in mediating inflammatory response. Thus, NOS generating NO from L-arginin or cyclooxygenase (COX) associated with synthesis of prostaglandins from arachidonic acid are main targets for blocking inflammation. According to previous studies, NO generated in a small amount by NOS which is expressed at a constant level in blood vessels and nerves plays an important role in maintaining homeostasis of normal bodies which induces neurotransmission and vasodilation. However, NO generated by induced NOS (iNOS) which is induced by various cytokines or external stimuli is known to cause cytotoxic or various inflammatory responses, and chronic inflammation is associated with the increase of iNOS activity (Appleton L. et al Adv. Phamacol., 35. 27-28. 1996).
According to another studies, cyclooxygenase includes two types of isoforms, of which cyclooxygenase-1 (COX-1) always resides in cells and synthesizes prostaglandins (PGs) necessary for cell protection, and COX-2 rapidly increases in cells in the inflammatory response and is known as playing a significant role in the inflammatory response.
Up to now, for anti-inflammatory drugs used for alleviating inflammation, nonsteroidal anti-inflammatory drugs include ibuprofen, flufenamic acid, indomethacin, etc., and steroidal anti-inflammatory drugs include prednisolone, dexamethasone, etc. Allantoin, glycyrrhetinic acid, and derivatives thereof are known to have anti-inflammatory effects. However, the development of raw materials having anti-inflammatory effects enough for consumers to feel the effects is still required.
Meanwhile, stem cells refer to cells having the potency to be differentiated into all types of cells constituting the body, such as nerves, blood, cartilage, etc., when required, while maintaining to be undifferentiated into specific cells. There are broadly two methods for obtaining these stem cells: first, obtaining from embryos generated from fertilized eggs (embryonic stem cells), and second, recollecting stem cells maintained in every part of the body in adults (adult stem cells). Embryonic stem cells and adult stem cells, although differ from each other in terms of functions, have the potency to be differentiated into various types of cells. Embryonic stem cells have excellent differentiating potency and long telomers, while having disadvantages of raising ethnical issues and having difficulty in obtaining a large amount of cells. In comparison, adult stem cells may obtain a large number of cells, while having disadvantages of posing risk of inflammation when transplanted into another body or having relatively low differentiating potency.
Despite the above disadvantages, adult stem cells are greatly stable for medical applications. Also, adipose-derived stem cell (ADSC), which can be readily obtained from suctioned lipid, does not raise ethical issues and can be readily obtained.
The primary ADSC is highly commercially applicable, but is not suitable for mass production of the culture media of stem cells due to slow growth rate and short lifespan.
In order to overcome the disadvantage, the present inventors prepared ADSC-T cell line in which T antigen of simian virus (SV 40) is introduced. The cell line has a threefold increase in proliferation rate and about 6-month prolonged cell lifespan, compared with primary ADSC line. Thus, the present invention supplements the disadvantage the primary ADSC line has with regard to mass production of culture media through the production of ADSC-T cell line in which T antigen is introduced into ADSC.
As a result of studies conducted by the present inventors to solve the issues on treatment, prevention, or improvement of anti-inflammation, skin-regeneration, whitening, antioxidant activity, or wound-healing, the present inventors established ADSC-T cell line in which T antigen is introduced into ADSC, and confirmed anti-inflammatory, skin-regenerative, whitening, antioxidant, or wound-healing effects of culture media of ADSC-T cell, and thereby completed the present invention.